Kit for prediction of pre-eclampsia

ABSTRACT

The present invention relates to a method of predicting pre-eclampsia (PE). The present invention also relates to a diagnostic kit for performing a method of predicting PE. In particular, the method determining the level of two or more markers selected from placenta growth factor (PlGF), plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor-2 (PAI-2) and leptin.

The present invention relates to a method of predicting pre-eclampsia(PE). The present invention also relates to a diagnostic kit forperforming a method of predicting PE.

PE is defined according to the guidelines of the International Societyfor the Study of Hypertension in Pregnancy (Davey et al., Am. J. ObstetGynecol; 158: 892-98, 1988). Gestational hypertension is defined as tworecordings of diastolic blood pressure of 90 mm Hg or higher at least 4h apart, and severe pressure of 110 mm Hg or higher at least 4 h apartor one recording of diastolic blood pressure of at least 120 mm Hg.Proteinuria is defined as excretion of 300 mg or more in 24 h or tworeadings of 2+ or higher on dipstick analysis of midstream or catheterurine specimens if no 24 h collection was available. Women areclassified as previously normotensive or with chronic hypertensionbefore 20 weeks' gestation. For previously normotensive women, PE isdefined as gestational hypertension with proteinuria and severe PE assevere gestational hypertension with proteinuria. For women with chronichypertension, superimposed PE is defined by the new development ofproteinuria. PE affects approximately 4% of all pregnancies and is aleading cause of maternal death in the UK. This disease, or the threatof onset, is the commonest cause of elective premature delivery,accounting for approximately 15% of all premature births. Themeasurement of blood pressure and testing for proteinuria in allpregnant women is carried out predominantly for the detection of PE.These procedures and the care of affected women and of the prematurechildren make considerable demands on healthcare resources. Accurateidentification of women at risk could dramatically reduce costs ofantenatal care.

Although, there is no widely used treatment for PE (other than prematuredelivery), we have recently reported a significant reduction in PE inhigh risk women given supplements of vitamin C and vitamin E (seeChappell et al., The Lancet, 354, 810-816, 1999). Risk was assessed by atest of relatively low sensitivity. More accurate and robustidentification of women at risk would target those women most likely tobenefit from this, or alternative, prophylactic therapies. Thoseidentified at lower risk could be provided with less intensive and lessexpensive antenatal care.

There is no widely accepted or accurate method for the early predictionof PE. Elevation of the blood pressure and detection of protein in theurine occur when the disease process is well established, as indicatedabove. Detection of an abnormality of the blood flow to the uterineartery by Doppler ultrasound in women who later develop PE has been ofsome predictive use but this abnormality has been found to be relativelynon-specific and for this reason has not been adopted in routineclinical practice.

Although some plasma/urine biochemical markers have been shown to beabnormal in the disease process, no single marker has proven to be ofadequate sensitivity for use as a predictive indicator. For example theuse of placenta growth factor (PlGF) alone as a predictive indicator ofPE has been proposed, but the predictive power of this marker could notbe determined with any certainty. For example, International patentapplication WO 98/28006 suggests detecting PlGF alone or in combinationwith vascular endothelial growth factor (VEGF) in order to predict thedevelopment of PE.

Furthermore, the effect of vitamin supplementation on the maternal bloodPAI-1/PAI-2 ratio has previously been published by our group (Chappellet al, 1999; Lancet, 354, 810-816) and others have documented raisedPAI-1/PAI-2 in established PE (Reith et al, 1993; British Journal ofObstetrics and Gynaecology 100, 370-4) and elevated PAI-1 in women whosubsequently developed PE (Halligan et al, 1994; British Journal ofObstetrics and Gynaecology 101, 488-92). PlGF has been shown to bereduced in women with established PE (Torry et al, 1998; AmericanJournal of Obstetrics and Gynaecology 179, 1539-44) and is suggested tobe low prior to the onset of the disease. Leptin has been found toincrease with gestation in normal pregnant women (Highman et al, 1998,American Journal of Obstetrics and Gynaecology 178, 1010-5), anobservation repeated by ourselves. Leptin has also been shown to riseeven further in established PE, the first report being published by Miseet al., Journal of Endocrinology and Metabolism, 83, 3225-9, 1998.Furthermore, Anim-Nyame et al., Hum. Reprod., 15, 2033-6, 2000,indicates that the elevation of leptin concentrations before PE isclinically evident. This finding is supported by Chappell et al., J.Soc. Gynecol. Invest., 213A, 2001, where it is also indicated thatvitamin supplementation reduces plasma leptin in women at risk of PE.

However, none of the prior art documents disclose a reliable, sensitiveand specific predictive test for PE.

It has now been found that a combination of markers provide the muchneeded predictive parameter for the desired early diagnosis of PE.

The present invention provides, a method of specific prediction ofpre-eclampsia (PE) comprising determining in a maternal sample the levelof two or more markers selected from placenta growth factor (PlGF),plasminogen activator inhibitor-1 (PAI-1), plasminogen activatorinhibitor-2 (PAI-2) and leptin.

It has been found that by measuring at least 2 of the markers mentionedabove that it is possible to determine with high specificity andsensitivity whether an individual is likely to develop PE. Specificityis defined as the proportion of true negatives (will not develop PE)identified as negatives in the method. Sensitivity is defined as theproportion of true positives (will develop PE) identified as positivesin the method. It is preferred that the method comprises measuring 3 ofthe markers, more preferably all four of the markers.

Preferably, the method of the present invention comprises determiningthe level of placenta growth factor (PlGF) with the level of one of thefollowing:

-   -   (i) plasminogen activator inhibitor-2 (PAI-2);    -   (ii) the ratio of plasminogen activator inhibitor-1 (PAI-1) to    -   (iii) leptin.

It has been found that these specific combinations are particularlyuseful for determining whether an individual is likely to develop PE.

The term “pre-eclampsia” as used herein is defined according to theguidelines of the International Society for the Study of Hypertension inPregnancy, as described above.

The term “specific prediction of pre-eclampsia” as used herein meansthat the method of the present invention is used to specifically predictthe development of PE. In particular, the method of the presentinvention enables one to determine whether an individual is likely todevelop PE.

The maternal sample is taken from a pregnant woman and can be any samplefrom which it is possible to measure the markers mentioned above.Preferably the sample is blood. The samples can be taken at any timefrom about 10 weeks gestation. Preferably the sample is taken at between12 and 38 weeks gestation, more preferably the samples are taken between20 and 36 weeks.

The term “placenta growth factor” (PlGF) refers to the free form foundin the individual. The amino acid sequence human PlGF is known (see NCBIProtein database, accession no. XP 040405). There are numerous methodsof detecting PlGF including the commercially available Quantikine HumanPlGF immunoassay from R&D Systems Inc.

The term “plasminogen activator inhibitor-1” (PAI-1) is a standard termused in the art and is clear to those skilled in the art. In particular,the sequence of the human form of PAI-1 is given in the NCBI Proteindatabase under accession no. AAA 60003. There are numerous methods ofdetecting PAI-1 including the commercially available Tint Elize PAI-1kit from Biopool International.

The term “plasminogen activator inhibitor-2” (PAI-2) is a standard termused in the art and is clear to those skilled in the art. In particular,the sequence of the human form of PAI-2 is given in the NCBI Proteindatabase under accession no. CAA 02099. There are numerous methods ofdetecting PAI-2 including the commercially available Tint Elize PAI-2kit from Biopool International.

The term “leptin” is a standard term well known to those skilled in theart. In particular, the amino acid sequence of a human form of leptin isgiven in the NCB1 Protein database under accession no. P41159. There arenumerous methods of detecting leptin, for example, the Quantikine, humanleptin immunoassay from R&D Systems Inc.

In a particularly preferred embodiment of the present invention, themethod of the present invention is performed by determining the level oftwo or more of the markers using the automatic DELFIA® system which isavailable from Wallac, Finland. Automatic DELFIA® is an automated systemspecifically designed and optimised for performing immunoassays and cantherefore be used to measure the levels of two or more of the markersused in the method of the present invention. The automatic DELFIA®systems measures the concentration of the markers using fluorescence andall four markers can be detected in a single well/sample.

We obtained samples of blood from pregnant women who were considered atrisk of PE on the basis of the uterine artery Doppler test or becausethey had had the disease in a previous pregnancy. Blood samples wereobtained from 20 weeks of pregnancy at intervals of 4 weeks untildelivery. We measured a selection of biochemical markers implicated inPE, including vitamin C, homocysteine, plasma lipids and 8-epiprostaglandin F_(2α) but none proved to be effective in prediction. Wefound that the ratio of plasminogen activator inhibitor-1 (PAI-1) andPAI-2 increased prior to the onset of the disease, whereas placentagrowth factor (PlGF) failed to show the pronounced rise normallyobserved in healthy pregnancies. Plasma leptin normally rises inpregnancy but we found that it increased to a much greater extent inwomen destined to develop PE. Combinations of these markers (see below)proved to be excellent in the sensitive and specific prediction ofsubsequent PE.

In testing the combinations described above it has been found that forpatients who will develop PE (i.e. the prediction is positive) there isno increase in the level of PlGF with gestation whereas PlGF normallyincreases with gestation. If the combination of markers PlGF and PAI-2is used, a positive prediction is given by the combined levels of PlGFand PAI-2 being less than normal.

Where the combination of markers PlGF and the ratio of PAI-1 to PAI-2 isused, a positive prediction is given by a combination of a reduced levelof PlGF and the ratio of PAI-1 to PAI-2 being greater than normal.

If the combination of the markers PlGF and leptin is used, a positiveprediction is given by the ratio of leptin to PlGF being greater thannormal.

In order to determine whether the level or ratio of the markers referredto above is greater than or less than normal, the normal level or ratioof the relevant population needs to be determined. The relevantpopulation may be defined based on, for example, ethnic background orany other characteristic that may affect normal levels of the markers.The relevant population for establishing the normal level or ratio ofthe markers is preferably selected on the basis of low risk for PE (i.e.no known risk marker for PE, such as previous PE, diabetes, priorhypertension etc.). Once the normal levels are known, the measuredlevels can be compared and the significance of the difference determinedusing standard statistical methods. If there is a statisticallysignificant difference between the measured level and the normal level,then there is a significant risk that the individual from whom thelevels have been measured will develop PE.

In a preferred embodiment of the present invention, the markers PlGF andPAI-2 may be combined using the algorithm:—d(log_(e)[PAI-2])+(log_(e)[PlGF])wherein d is a constant in the range of about 0.03 to 48.6. Preferably dis in the range of 0.072 to 7.6, more preferably in the range of 0.0336to 2.2. Most preferably d is 0.75 or 1. Alternatively markers PlGF andPAI-2 may be combined using the algorithm:—[PAI-2]^(d)*[PlGF]wherein d is as defined above. The sign “*” is used as the sign formultiplication.

In a particularly preferred embodiment, d is 1 and the previousalgorithm can be written as [PAI-2]*[PlGF]. Using this algorithm, andassuming the concentration of PAI-2 is measured as ng/ml and theconcentration of PlGF is measure as pg/ml, it has been found that if thevalue obtained using the algorithm is <35,000 that the sensitivity andspecificity of predicting PE is 67% and 100%, respectively. If the valueobtained using the algorithm is <50,000 that the sensitivity andspecificity of predicting PE is 80% and 94%, respectively (see Table 10below).

In a further preferred embodiment of the present invention, the markersPlGF and the ratio of PAI-1/PAI-2 may be combined using the algorithm:—(log_(e)[PlGF])−(g*{PAI-1/PAI-2 ratio})wherein g is a constant in the range of about −19.4 to 3.6. Preferably gis in the range of 0.655 to 15.5, more preferably 1.37 to 7.0. In aparticularly preferred embodiment g is 3.0. Using the algorithm when gis 3.0, and assuming the concentration of PlGF is measured as pg/ml, ithas been found that if the value obtained using the algorithm is <4.5that the sensitivity and specificity of predicting PE is 53% and 100%,respectively. If the value obtained using the algorithm is <5 thesensitivity and specificity of predicting PE is 80% and 88%,respectively (see Table 4 below).

It has also been found that by measuring the leptin/PlGF ratio, when theleptin concentration is in ng/ml and PlGF concentration is pg/ml, avalue of >0.1 provides a method of predicting PE with 67% sensitivityand 100% specificity. When the value is >0.05, the method of predictingPE has 80% sensitivity and 88% specificity.

It can be seen that the level of sensitivity and specificity can bealtered by altering the threshold level. In some situations, e.g. whenscreening large numbers of women at low risk of PE, it is important tohave high specificity. In other situations, it may be important to havea balance between high sensitivity and specificity, e.g. whenconsidering individual women at high risk of PE a balance between highsensitivity and specificity is needed.

The present invention offers many benefits. In addition to facilitatingaccurate targeting of interventions e.g. vitamin supplements,considerable saving on health care resources can be expected due tostratification of antenatal care and reduced neonatal special carecosts. In the research and development area, identification of high riskpatients will greatly facilitate future clinical trials. At present dueto inadequate methods of prediction, large numbers of pregnant womenunnecessarily receive interventions in clinical trials.

The method the present invention may be performed in conjunction withother tests for diagnostic indicators, such as blood pressure, level ofuric acid etc.

The method of the present invention may also be used in order to monitorthe efficiency of a prophylactic treatment for preventing thedevelopment of PE, wherein a reduction in the risk of developing PE willbe indicative of the prophylactic treatment working.

More than twenty biochemical markers have been shown previously to beassociated with established PE and there would be no logical priorreason for choosing PAI-1, PAI-2, PlGF and leptin in any prospectivelongitudinal study for assessment of use as predictive indicators.Moreover very few groups have evaluated any individual markerprospectively in the same women from whom samples were taken atintervals throughout their pregnancy. Importantly none has measured thedifferent markers in the same women, unlike in the present application.

Once a value has been obtained using one of the algorithms mentionedabove, the log-odds of the individual developing PE can be calculatedusing the formula:y=a+bxwherein y is the log-odds of the individual developing PE, x is thevalue obtained using one of the algorithms and a and b are constants(values provided later) derived from logistic regression analysis of ourpreviously acquired data set adjusted on the assumption of 4% prevalenceof PE in the population. This approach, known as logistic regression, iswidely used in clinical research.

In order to demonstrate how the formula can be used to determinelog-odds of an individual developing PE, the following informationdemonstrates how it is possible to determine the desired values of a andb so that a log-odds value can be obtained having any desired confidenceinterval (CI).

The following prediction formulae are calculated based on the sample ofpre-eclamptics and controls analysed at 24 weeks. The formulae give thelog-odds of PE for any given value of the predictor. The probability isjust exp(log-odds)/(1+exp(log-odds)) (exp means the inverse function ofthe natural logarithm).

All values are given corrected for a prevalence of 4%, log-odds of4%=log(4/96)=−3.18. To convert to a different prevalence, say 20%, firstwork out the new log-odds=log(20/80)=−1.39. The difference is−3.18−(−1.39)=1.79

The value of the constant “a” must be increased by this amount.

The value of “b” is unchanged.

The best values of “a” for use with algorithmlog_(e)[PlGF]−3*(PAI-1/PAI-2), giving the highest CI is 23.042. However,the value for “a” with a CI of 75%, 95% or 99% is:

75% limits: 9.314 to 36.771

95% limits: −0.348 to 46.432

99% limits: −7.697 to 53.782

The best value of “b” for use with algorithmlog_(e)[PlGF]−3*(PAI-1/PAI-2), giving the highest CI is −5.223. However,the value for “b” with a CI of 75%, 95% or 99% is:

75% limits: −7.940 to −5.620

95% limits: −9.852 to −3.708

99% limits: −11.306 to −2.254

The best value of “a” for use with the algorithm leptin/PlGF ratio is−5.801. However, the value of “a” with a CI of 75%, 95% or 99% is:

75% limits: −6.895 to −4.707

95% limits: −7.665 to −3.937

99% limits: −8.251 to −3.351

The best value of “b” for use with the algorithm leptin/PlGF ratio is42.197. However, the value of “b” with a CI of 75%, 95% or 99% is:

75% limits: 22.393 to 58.948

95% limits: 8.455 to 72.886

99% limits: −2.147 to 83.489

The best value of “a” for use with the algorithm [PAI-2]*[PlGF] is−0.919. However, the value of “a” with a CI of 75%, 95% or 99% is:

75% limits: −1.923 to 0.084

95% limits: −2.630 to 0.791

99% limits: −3.167 to 1.328

The best value of “b” for use with the algorithm [PAI-2]*[PlGF] is0.000. However, the value of “b” with a CI of 75%, 95% or 99% is:

75% limits: −0.000 to −3.114

95% limits: −0.000 to −3.114

99% limits: −0.000 to −3.114

It is therefore possible for those skilled in the art to determine thelog-odds of a patient developing PE with any desired CI based on theinformation given herein and by using standard statistical analysis.

The present invention also provides a diagnostic kit for performing themethod of the present invention. The kit comprises reagents required todetermine the level of the markers being measured. Suitable agents forassaying for the markers include enzyme linked immunoassay reagents, RIAreagents and reagents for Western blotting.

The present invention is now described by way of example only, withreference to the following figures.

FIG. 1 shows an ROC (Receiver Operation Characteristic) curve for theprediction of PE, based on the formula (log_(e)[PlGF])−(3.0*{PAI-1/PAI-2ratio}) from data at 24 weeks' gestation.

FIG. 2 shows the level of PAI-2 variation during the gestation period,wherein (▪) is low risk women, (▴) is women who subsequently developedPE, and (●) is women who did not develop PE but delivered small forgestational age (SGA) infants.

FIG. 3 shows the level of Leptin variation during the gestation period,wherein (▪) is low risk women, (▴) is women who subsequently developedPE, and (●) is women who did not develop PE but delivered small forgestational age (SGA) infants.

FIG. 4 shows the level of PlGF variation during the gestation period,wherein (▪) is low risk women, (▴) is women who subsequently developedPE, and (●) is women who did not develop PE but delivered small forgestational age (SGA) infants.

FIG. 5 shows the level of PAI-1 variation during the gestation period,wherein (▪) is low risk women, (▴) is women who subsequently developedPE, and (●) is women who did not develop PE but delivered small forgestational age (SGA) infants.

FIG. 6 shows the level of PAI-1/PAI-2 ratio variation during thegestation period, wherein (▪) is low risk women, (▴) is women whosubsequently developed PE, and (●) is women who did not develop PE butdelivered small for gestational age (SGA) infants.

FIG. 7 shows the level of Leptin/PlGF ratio variation during thegestation period.

FIG. 8 shows the level of (log_(e)[PlGF])−(3.0*{PAI-1/PAI-2 ratio})variation during the gestation period.

FIG. 9 shows the level of (log_(e)[PAI-2])+(log_(e)[PlGF]) variationduring the gestation period.

EXAMPLES Example 1

The method of the present invention is preferably carried out at the20th week of pregnancy or later e.g. at 24 weeks.

Briefly, the method of the present invention is performed by taking 5mls of venous blood from a pregnant woman into a vacutainer with eithertrisodium citrate or 0.5M EDTA as anticoagulant. The plasma is decantedafter centrifugation and stored at −20° C. until assay. Use may be madeof commercially available assays such as the following: Assays forleptin (Quantikine, Human Leptin immunoassay, immunoassay R& D systemsInc, Minneapolis Minn. 55413, USA); PlGF (Quantikine Human PlGFimmunoassay R& D systems Inc, as above); PAI-1 (TintElize PAI-1, BiopoolInternational, Umea, Sweden or Ventura Calif. 93003, USA) and PAI-2(TintElize PAI-2, Biopool International, as above). The assays areperformed according to the manufacturer's instructions. The followingare calculated from the plasma concentrations obtained in the assays:(log_(e)[PlGF])−(3.0*PAI-1/PAI-2 ratio)  1.0.75(log_(e)[PAI-2])+(log_(e)[PlGF])  2.leptin/PlGF  3.

The number calculated in 1, 2 or 3 (referred to below as “x”) is thenentered on specially designed software (provided) in the equationy=a+bxwhere y is the calculated log-odds of the patient developing PE and aand b are constants (values provided later) derived from logisticregression analysis of our data set adjusted on the assumption of 4%prevalence of PE in the population and x is the calculated value from 1,2 or 3. This approach, known as logistic regression, is widely used inclinical research. We claim novelty for establishing appropriate valuesfor a and b in this context.

The probability (0-100%) of developing PE for each of the three tests isgiven by exp [y/(1+y)]*100%. This value can be adjusted for populationprevalence of PE or by risk for an individual patient.

The method of testing for prediction of PE involves the simultaneousmeasurement of PAI-1, PAI-2, PlGF and leptin in ‘kit’ form. Each assayis currently based on a colorimetric test e.g. an enzyme linkedimmunoabsorbent assay, ELISA, in which intensity of colour developmentin a test ‘well’ is proportional to the concentration of marker present.The kit involves four wells, one specific for each marker and the tester(hospital biochemist) adds a known volume of the pregnant woman's bloodplasma to each well. The colours are then assessed simultaneously on acolour density scanner. These scanners are available in all routinehospital laboratories. The result for each marker (obtained on the printout from the scanner) is then typed into a specially designed computerprogram. For each of the algorithms described above the program computesa single value. This value can be compared to the limits of the normalrange provided in Table 4 below.

Depending on this value, the woman's % risk (0-100%) is assessed anddetermined

As indicated previously, it is particularly preferred that the method ofthe present invention is performed using the automatic DELFIA® system.

Algorithm Development

-   -   In devising algorithms for the combination of the specified        markers, the best value was obtained using        (log_(e)[PlGF])−(3.0*{PAI-1/PAI-2 ratio}). At 24 weeks        gestation, the area under the ROC curve was 0.96 (95% CI        0.88-1.99). A perfect test would give an area of 1 whilst a test        no better than chance would give an area of 0.5. This formula        also worked well for samples tested at earlier and later weeks        of gestation, although to be of clinical use the earlier the        risk can be assessed, the more useful will be the test. Areas        under the curve at the different gestations tested are given        below in Table 1 and shown graphically for 24 weeks gestation in        the FIG. 1.

TABLE 1 Gestation ROC area 95% CI 20 weeks 0.81 0.63-0.98 24 weeks 0.960.88-1.00 28 weeks 0.91 0.78-1.00 32 weeks 0.96 0.90-1.00 36 weeks 0.990.97-1.00

We have also found that combination of PAI-2 and PlGF gives an almostequally good prediction of risk using the algorithm0.75(log_(e)[PAI-2])+(log_(e)[PlGF]). See Table 2.

TABLE 2 Gestation ROC area under curve 95% confidence interval 20 weeks0.80 0.60-1.00 24 weeks 0.88 0.74-1.00 28 weeks 0.91 0.77-1.00 32 weeks0.94 0.86-1.00 36 weeks 0.97 0.91-1.00

Additionally we found that a combination the ratio of Leptin/PlGF is agood predictive indicator of PE (see Table 3).

TABLE 3 Gestation ROC area under curve 95% confidence interval 20 weeks0.78 0.59-0.98 24 weeks 0.87 0.74-1.00 28 weeks 0.80 0.60-1.00 32 weeks0.96 0.90-1.00 36 weeks 0.90 0.75-1.00

An additional value of these prediction tests lies in their poorpredictive value for the later development of growth retardation.Several markers, particularly those synthesized in placental tissue, aresimilarly raised in PE and in pregnancies associated with fetal growthretardation but uncomplicated by PE. Neither of the combinations ofmarkers we have used were predictive of growth retardation i.e. they arespecific for PE.

The following Tables show typical values of the markers and markerratios and values obtained from the corresponding algorithms givenabove.

TABLE 4 Normal Ranges in healthy women with normal pregnancy outcomes.Normal Range (90% Marker Median Reference Range) PlGF pg/ml 586  292 to1177 PAI-1 ng/ml 40.0 25.4 to 63.0 PAI-2 ng/ml 169  78 to 363PAI-1/PAI-2 0.24 0.10 to 0.55 Leptin ng/ml 18.7  8.4 to 42.0 Log ePlGF −(3.0 × {PAI-1/PAI-2} ratio 5.57 4.71 to 6.43 Leptin/PlGF 0.030 0.013 to0.069 0.75(logPAI-2) + (logPlGF) 10.20  9.30 to 11.00

TABLE 5 PE-ranges in high risk women who later develop PE Normal Range(90% Marker Median Reference Range) PlGF pg/ml 221  54 to 910 PAI-1ng/ml 39.8 23.5 to 67.5 PAI-2 ng/ml 103.0  49.4 to 214.6 PAI-1/PAI-20.387 0.180 to 0.830 Leptin ng/ml 30.7 14.9 to 63.2 Log_(e)PlGF − (3.0 ×{PAI-1/PAI-2} ratio 4.01 2.36 to 5.67 Leptin/PlGF 0.124 0.020 to 0.7640.75(log_(e) PAI-2) + (log_(e) PlGF) 8.80  7.20 to 10.40

TABLE 6 Values for a and b in algorithms 1 to 3. Equation a bLog_(e)PlGF − (3.0 × {PAI-1/PAI-2} ratio 28.1 −5.65 Leptin/PlGF 6.562.31 0.75(log_(e) PAI-2) + (log_(e) PlGF) 24.9 2.62

The variation in the ratio of leptin to PlGF for controls and women wholater developed PE is shown in FIG. 7. The variation in PlGF andPAI-1/PAI-2 ratio as determined using algorithmlog_(e)[PlGF]−3*(PAI-1/PAI-2) for controls and women who later developedPE is shown in FIG. 8. The variation in PAI-2 and PlGF levels asdetermined using algorithm 0.75 (log_(e)[PAI-2])+log_(e)[PlGF] forcontrols and women who later developed PE is shown in FIG. 9.

Example 2 Methods

Subjects. Subjects were recruited with local ethical committee approvalfrom St Thomas' Hospital and Chelsea and Westminster Hospital, London,UK.

High risk women were identified by PE requiring delivery before 37weeks' gestation in the preceding pregnancy or by abnormal uterineartery Doppler FVW (defined as a resistance index ≧95^(th) centile forgestation or the presence of an early diastolic notch). The study groupwere drawn from the placebo arm of a randomized clinical trial ofantioxidant supplementation. 1512 women were screened at 18-22 weeks andat 24 weeks gestation for persistent abnormalities of the Doppler FVW. Atotal of 160 women participated in the clinical trial of antioxidantsuntil delivery. Of the 81 high-risk women reported in the present studyfrom the placebo arm, 60 women entered the study on the basis ofabnormal Doppler FVW and 21 on the basis of PE in the previouspregnancy. The 81 women were followed longitudinally with blood samplingat 4 weekly intervals. Data from the women who developed either PE withor without SGA (PE, n=21) or who delivered small for gestational age(SGA, n=17) infants without PE are reported. Of the women who developedPE, six had essential hypertension (five were taking methydopa at thetime of recruitment) and one had antiphospholipid syndrome. Five womenwere taking aspirin; this was not an exclusion criterion for the trial.Gestational Pre-eclampsia is defined by the International Society forthe Study of Hypertension in Pregnancy guidelines (Am. J. ObstetGynecol., 158: 892-98, 1988), which describes PE as gestationalhypertension with superimposed PE. Gestational hypertension was definedas two recordings of diastolic blood pressure ≧90 mmHg≧4 hours apart andsevere gestational hypertension as two recordings of diastolic bloodpressure ≧110 mmHg≧4 hours apart or one recording of diastolic bloodpressure ≧120 mmHg. Proteinuria was defined as ≧300 mg/24 hrs or tworeadings of ≧2+ on dipstick analysis of mid-stream or catheter urinespecimens if no 24 hour collection was available. SGA infants weredefined as those ≦10^(th) centile for gestation and gender, correctedfor maternal height, weight, parity and ethnicity using centile charts(Lancet et al., 339: 283-287, 1992).

Low risk women All women attending the hospital antenatal clinics forroutine care during the trial recruitment period who consented to thestudy and who, on screening, had a normal Doppler FVW and no otherco-existing disease or risk markers were invited to participate. 33consented and 1 failed to finish the study; data are presented from the27 women who delivered infants of appropriate size for gestational age(AGA). Since SGA infants delivered by low risk women (with normaluterine artery Doppler FVW) are more likely to be ‘normally’ small thanto be growth restricted, pregnancies associated with SGA in this groupwere not included in the SGA group.

Blood sampling. Venous blood was drawn from an uncuffed arm into tubeswith appropriate additions for each of the factors (also referred toherein as markers) assayed. Samples were placed immediately on ice andcentrifuged within 3 hrs. Supernatants were stored at −70° C. prior toassay.

Analysis of Biochemical Markers

Indices of Antioxidant Status and Oxidative Stress

Samples for assay of ascorbic acid and α tocopherol were stored in 2%metaphosphoric acid. Ascorbic acid and uric acid were determined byreverse phase high pressure liquid chromatography (HPLC) (Pediatr Res etal., 36: 487-93, 1994) (ascorbic acid; lower limit of detection 5 nM;intra-assay coefficient of variation [CV] 2.2%; inter-assay CV 3.5%;uric acid; lower limit of detection 5 nM; intra-assay CV 2.6%,inter-assay CV 3.8%). α-tocopherol was assayed by reverse phase HPLC(Br. J. Nutr et al., 63: 631-8, 1990) (lower limit of detection 10 nM;intra-assay CV 2.1%; inter-assay CV 3.9%). Due to sample losses ofmethodological origin the isoprostane 8-epi-PGF_(2α) (a marker of lipidperoxidation) was not determined in all women, but was assessed inavailable samples from 21 low risk, 13 SGA and 17 pre-eclamptic women aspreviously described (J Chromatogr B. Biomedical Applications., 667:199-208, 1995), by gas chromatography-mass spectrometry. Previousstudies from our laboratory indicated that these numbers would provideadequate power to reveal significant differences between groups.

Indices of placental insufficiency Plasminogen activator inhibitor(PAI-2) was determined by ELISA (Tintelize, Biopool International,Sweden; lower limit of detection 6 ng/ml; intra-assay CV 3.7%;inter-assay CV 3.0%). Serum leptin was evaluated by RIA using¹²⁵labelled human leptin (LINCO Research, Inc, Missouri, USA; lowerlimit of detection 0.5 ng/ml; intra assay CV 4.5%; inter-assay CV 4.9%).PlGF was evaluated by ELISA (R&D systems, Abingdon, UK; lower limit ofdetection 7 pg/ml; intra assay CV 5.6%-7.0%; inter-assay CV10.9%-11.8%).

Index of endothelial function. Plasminogen activator inhibitor-1 (PAI-1)was determined by ELISA (Tintelize, Biopool International, Sweden; lowerlimit of detection 0.5 ng/ml; intra-assay CV 3.3%; inter-assay CV 2.9%).

Lipids Serum triglycerides and total cholesterol were measured byenzymatic colorimetric tests (UNIMATE 5 TRIG and UNIMATE 5 CHOLrespectively, Roche/BCl, Lewes, Sussex, UK). HDL-cholesterol wasdetermined by detergent based isolation and enzyme linked colorimetricdetection (DIRECT HDL CHOLESTEROL, Randox laboratories, Co Antrim,Northern Ireland). LDL-cholesterol was estimated by calculation fromtriglycerides and HDL cholesterol. Apo A-1 and Apo B were evaluated byimmunoturbidimetry (Dade/Behring, Milton Keynes, UK).

Statistical Analysis

Data were analysed in Stata 6.0 (StataCorp, College Station, Tex.)Summary scores (mean of 2 or more measurements made in weeks 20-36) werecalculated for each biochemical marker (Matthews et al., Br Med. J.,300: 230-5, 1990). Log transformations & geometric means were used for8-epi-PGF_(2α), leptin, PAI-1, PAI-2, PAI-1/PAI-2 ratio, triglycerides,vitamin E/cholesterol ratio and uric acid. As PAI-1 changed considerablywith gestation, a 2-way interaction between time and outcome were fittedwith Generalised Estimating Equations (GEE). (Biometrika et al., 73:13-22, 1986) GEE was also used to estimate the impact of race(Caucasian/European vs. African/Caribbean) and parity.

Markers showing significant differences (8-epi PGF2α, HDL Cholesterol,Uric acid, PAI-1/PAI-2 ratio, leptin and PlGF) were considered aspossible predictors of PE at 20 and 24 weeks. Areas derived fromReceiver Operation Characteristic (ROC) curves were used to assess theirusefulness. Multiple logistic regression was use to develop threecombined predictive indices (details available on request). Sensitivityand specificity were calculated for appropriate cut-points. A smoothedROC curve (Stata Technical Bulletin., 2000; 52: sg120) is given for thebest index.

Percentage differences from the reference group are given with 95%confidence intervals (CI) using robust standard errors (Biometrika etal., 73: 57-64, 1988). Significance at the 5% level is claimed when theCI excludes no effect (0% or ROC area 0.5).

Results

Study entry details are given in Table 7 and perinatal characteristicsin Table 8. There were 45% (95% CI 21 to 69%) more women of African orCaribbean origin in the PE group than in the low risk group; no otherdifferences were significant.

Longitudinal Profile of Biochemical Markers

Some women delivered before the last (36 week) sample. There were a fewadditional omissions due to failure to attend the clinic and loss ofsamples for methodological reasons. Biochemical markers other than8-epi-PGF_(2α) (as detailed above) were measured on at least fouroccasions for the majority of women (66%-84%, mean 78% of women;depending on marker).

Indices of antioxidant status and oxidative stress Plasma ascorbic acidconcentrations were decreased in both the SGA (−39%; CI −61% to −17%)and PE groups (−30%; CI −50% to −11%) compared to low risk women.Differences between SGA and PE groups were not significant. Plasmaα-tocopherol concentrations corrected for cholesterol showed a smallrise over gestation in the low risk women, but no significantdifferences were observed between groups. Summary scores for plasma8-epi-PGF_(2α) concentrations showed a trend towards higher values inthe PE group (mean difference 51%; CI −1% to 131%) compared to the lowrisk women. A less pronounced trend was also observed in the SGA group(−41%; CI −6% to 114%). Uric acid concentrations increased withgestation in all groups but the rise in the PE group was greater than inlow risk women (21%; CI 8% to 36%) or in the SGA group (difference 19%,CI 4% to 37%).

Indices of placental insufficiency. Compared with low risk women, thePAI-2 concentration was lower in both the SGA (−28%; CI −41% to −11%)and PE groups (−43%; CI −55% to −26%) but the difference between thelatter groups was not significant (see FIG. 2). The serum leptinconcentration was significantly higher in the PE group compared with SGA(92%; CI 39% to 165%) or low risk groups (74%, CI 21% to 135%) andvalues in the SGA and low-risk groups were similar (see FIG. 3). Thesedifferences remained significant after correction for BMI. PlGF in thelow risk women rose and then fell with gestational age (see FIG. 4).This profile was blunted in the SGA group (−35%;

CI −57% to −3%) and almost abolished in the PE group (compared withlow-risk −63%; CI −77% to −40%; compared with SGA −42%; CI −67% to +1%).

Index of endothelial function and PAI-1/PAI-2 ratio. PAI-1 increasedwith gestation in all groups. Plasma concentrations were significantlyhigher in the PE (13%; CI 2% to 25%) compared to low risk group (seeFIG. 5). The PAI-1/PAI-2 ratio fell in the low-risk women by −26% (CI−41% to −8%) over gestation, showed no overall change in the SGA groupbut increased in the PE group by 62% (CI 17% to 122%). Compared with lowrisk women the PAI-1/PAI-2 ratio was 45% higher in the SGA (CI 15% to82%) and 85% higher in the PE (CI 44% to 139%) groups, the differencebeing 28% (CI −3% to 70%) (see FIG. 6).

Lipids Serum triglyceride concentrations increased with gestation, beinghighest in the PE group (difference from low risk women 29%, CI 2% to62%). Serum HDL-cholesterol was 13% lower in the PE group than in lowrisk women (CI −24% to −2%). No differences between groups were observedin total and LDL-cholesterol, apo-A1 or apo-B concentrations (data notshown).

Biochemical Indices and Blood Pressure for Prediction of Pre-Eclampsia

Table 9 gives ROC areas for the prediction of PE at 20 and 24 weeks'gestation using six markers identified as potential predictiveindicators. At 20 weeks' gestation HDL cholesterol, PAI-1/PAI-2 ratio,leptin and PlGF were able to distinguish PE from low risk women (ROCareas significantly >0.5) and HDL cholesterol and leptin distinguishedsubsequent PE from SGA. At 24 weeks' gestation, PAI-1/PAI-2 ratio,leptin and PlGF gave ROC values >0.75 (where chance=0.5 and perfectvalue=1.00) for the PE group compared to low risk, and uric acid wasmarginally significant. Leptin, PlGF and uric acid distinguished betweenthe PE and SGA groups. A series of logistic regression analyses led tothree algorithms with ROC values 0.89 for the prediction of PE at 24weeks and ≧0.80 at 20 weeks (Table 9B) in comparison with the low riskwomen. These algorithms also distinguished significantly the PE from theSGA group at 24 weeks' gestation. An example of a ROC curve for one ofthese algorithms (log_(e)[PlGF])−(3.0*(PAI-1/PAI-2 ratio) at 24 weeksgestation is shown in FIG. 1.

Blood pressure (mean arterial, systolic and diastolic) at booking and at20 weeks was highly predictive of subsequent PE (e.g. booking blood meanarterial pressure; ROC area % PE vs LR; 0.79, CI 0.66 to 0.92; systolicBP 0.78, CI 0.65 to 0.91 and diastolic BP 0.80, CI 0.68 to 0.98), butthese data are strongly influenced by 6 women with pre-existinghypertension in the high risk group, a known risk marker for PE.

There was no statistical evidence that any of the three main indices orany combination was affected by parity, or that values for prediction ofPE were different between ethnic groups. Two threshold values chosen tomaximise a) sensitivity and b) specificity were defined for eachindicator. Values at 24 weeks' gestation, compared to the low risk groupare given in Table 10.

DISCUSSION

The data provided herein our knowledge, provides the most comprehensivelongitudinal study to date of biochemical indices of the disease in theblood of women destined to develop PE. Previous prospective longitudinalinvestigations have focussed on evaluation of single biochemicalmarkers, often in fewer subjects and have not compared the profiles inPE with women who delivered small for gestational age infants, but whodid not develop PE. The present data, in documenting substantivedifferences between profiles of the markers in pre-eclamptic pregnanciesand those in SGA deliveries uncomplicated by the disease has providedinteresting insight into the aetiology of the condition. Additionallycombinations of the markers identified, are useful in the prediction ofPE. A test that distinguishes subsequent PE from pregnanciescharacterised by fetal growth restriction alone is clinically useful,particularly as an adjunct to Doppler FVW analysis. Such discriminationearly in pregnancy would alert the obstetrician and pregnant woman toheightened surveillance of the symptoms of PE and permit interventionfor the prevention of PE should a clinically proven intervention becomeavailable e.g. vitamin C and E or calcium supplementation.

Whilst we recognize there are limitations in the use of the birthweightcentile as a surrogate marker of fetal growth restriction, importantdifferences from both low risk and PE groups were observed in the SGAgroup and these have provided valuable mechanistic insight. The majorityof high-risk women were recruited on the basis of an abnormal DopplerFVW, indicative of failed trophoblast invasion and high uteroplacentalresistance. Plasma concentrations of ascorbic acid in the healthycontrols were stable over gestation. In comparison, maternalconcentrations of ascorbic acid were significantly low in both SGA andPE groups throughout pregnancy. This would concur with the hypothesisthat poor uteroplacental perfusion predisposes to an increase inplacental free radical synthesis and, thereby to maternal oxidativestress. Without knowledge of daily intake, a contribution from lowerdietary vitamin C cannot be discounted, although the increased rate ofconsumption of ascorbate documented in the plasma of woman with PE wouldindicate that excessive metabolic consumption of vitamin C is the morelikely explanation. The trend toward elevated concentrations of theisoprostane 8-epi-PGF_(2α) in the PE group (p=0.055), despiteconsiderable scatter in the data, is indicative of oxidative stress.8-epi-PGF_(2α), a marker of lipid peroxidation, is present in thepre-eclamptic placenta and is variably reported to be increased (Clin.Sci et al., 91: 711-18, 1996) or be normal (Br. J. Obstet. Gynaecol etal., 105: 1195-99, 1998) in the maternal plasma in affected women.Further evidence for oxidative stress lies in the early increase in thePE group, but not in the SGA group of uric acid, a product of thexanthine/xanthine oxidase pathway. Reduced renal clearance of uric acidcould also lead to raised plasma concentrations in established PE, butthis is unlikely to explain the rise observed prior to clinicalmanifestation of the disease.

Since ascorbic acid concentrations were low in both PE and SGA groups, aspecific role for oxidative stress in the origin of PE might bequestioned. However, Hubel et al., pp 453-486, 1999, have suggested thatthe women who develop PE may be more likely to synthesise damaging lipidperoxides i.e. develop an exaggerated response to the oxidant burden, atheory supported by the much greater trend towards higher values of8-epi-PGF_(2α) in the PE group. This may arise from the wellcharacterized maternal dyslidipidaemia in PE, includinghypertriglyceridaemia (Hubel et al., pp 453-486, 1999) (which occurredas early as 20 weeks' gestation in this study), raised free fatty acidconcentrations and decreased LDL particle size which together maycontribute to the formation of damaging lipid peroxides and subsequentendothelial cell activation. Other risk markers including diabetes andessential hypertension, with associated microvascular dysfunction, mayalso influence the circulatory response to a pro-oxidant burden.

The lipid profile in this study showed a specific rise in the serumtriglyceride concentration in the women who developed PE. Elevation oftriglcyerides has previously been described at 10 weeks gestation inwomen who later develop PE our study (Hubel et al., pp 453-486, 1999);confirms an early elevation and may suggest that triglycerides play animportant pathophysiological role. Previous studies have documented afall in HDL cholesterol in women with established PE (Hubel et al., pp453-486, 1999); in the present study HDL was selectively reduced inwomen who later developed the disease, again implicating dyslipidaemiain the disease process. There was no difference in the LDL cholesterolconcentrations, but it is recognized that the properties rather than theabsolute concentrations of LDLs are altered in PE.

The abnormal concentration of leptin is likely to reflect placentalinsufficiency. The substantial increase of maternal blood leptinconcentrations in normal pregnancy is generally ascribed to placentalsynthesis since leptin is synthesised in the placenta (Ashworth et al.,5: 18-24, 2000) although leptin synthesis by maternal adipocytes islikely to contribute. Previous studies have reported a further increasein serum leptin concentrations in women with PE possibly reflectingplacental hypoxia (Mise et al., J. Clin. Endocrinol. Metab., 83:3225-29, 1998). The selective early elevation of leptin concentrationsin this study in the women who later developed PE may indicate a role asa prognostic indicator. Early elevation of leptin in women destined todevelop PE has recently been described (Anim-Nyame et al., Hum. Reprod.,15: 2003-6, 2000), although no other high risk groups were investigated.Of added interest in the present study was the finding that serum leptinwas no different in the healthy pregnant women and those who deliveredSGA infants. Correction for BMI (body mass index) did not alter thedifferences observed. If the rise in leptin in women who developed PEresults from hypoxia then this must be presumed to be less pronounced inthe SGA group. Alternatively, leptin synthesis is stimulated bycytokines, recognized to contribute to the inflammatory state associatedwith PE. An increase in the serum leptin concentration may alsocontribute to an inflammatory response and vascular dysfunction, as thepeptide itself has pro-inflammatory properties.

Whereas leptin was selectively increased, another marker of placentalinsufficiency, PlGF was substantially and selectively reduced in womenwho later developed PE, also holding promise for this angiogenic markeras a potential predictive indicator. This agrees with previous crosssectional studies reporting that low plasma PlGF concentrations arecharacteristic of PE (Torry et al., Am. J. Obstet. Gynecol., 179: 1539,1998) and our study confirms a recent report by Tidwell et al., Am. J.Obstet. Gynecol., 184: 1267-1272, 2001 which has shown an early decreasein plasma PlGF in women who subsequently developed PE. Another report(Livingston et al., Am. J. Obstet. Gynecol., 184: 1218-1220, 2001) inwhich samples were taken twice, once at 20 weeks and upon diagnosis ofPE has shown no difference in PlGF at 20 weeks gestation. In our studythe blunted PlGF concentrations whilst markedly more abnormal at 24weeks gestation were also modestly, but significantly reduced at 20weeks' gestation. In contrast to leptin, lowered oxygen tensiondown-regulates PlGF (Ahmed et al., Placenta., 21; S16-24, 2000) and mayprovide an explanation for failure of the normal increase. Theconsequences of reduced PlGF may be deleterious, potentially leading topoor trophoblast proliferation, reduced protection against apoptosis andcompromised vascular development.

The maternal concentration of PAI-2, also synthesized in placentaltrophoblast was less selective in discriminating pre-eclampticpregnancies, being reduced in both the PE and SGA groups, as previouslyreported (Lindoff et al., Am. J. Obstet. Gynecol., 171: 60-64, 1994).PAI-1, the only endothelial marker studied was elevated, particularlytowards the end of pregnancy in the pre-eclamptic group. As PAI-2 fallsand PAI-1 increases, as previously reported in established PE (Halliganet al., Br. J. Obstet. Gynaecol., 101: 488-92, 1994), the PAI-1/PAI-2ratio increases (Reith et al., Br. J. Obstet. Gynaecol., 100: 370-74,1993). We report here that an abnormally raised PAI-1/PAI-2 ratio alsopredates the onset of PE.

This study offered the unique opportunity of evaluating the potentialvalue of various combinations of markers in discrimination andprediction of pre-eclamptic pregnancies. No previous study hassimultaneously assessed a wide range of relevant biochemical indices.Individually, six of the markers showed significance for prediction ofPE at 20 weeks' gestation and serum leptin and HDL cholesterol showedgood discrimination between pre-eclamptic and SGA groups. PlGF showedgreatest discrimination at 24 weeks. Three specific combinations of themarkers studied showed they can be used to predict PE; a combination ofPlGF and the PAI-1:PAI-2 ratio, a combination of PAI-2 and PlGF and thecombination leptin:PlGF ratio. When measured at 24 weeks thesecombinations predicted the later development of PE with the potentialfor high specificity if used as a screening test, and high sensitivityif used in high-risk women. Prediction at 20 weeks was almost as high.These data compare favourably with values for other potential screeningtests for PE (Friedman S A et al., and Lindheimer M D. Prediction andDifferential Diagnosis in Chesley's Hypertensive Disorders in Pregnancy.Ed: Lindheimer M D, Roberts J M. Cunningham G. Appleton & Lang,Connecticut, USA. pp 201-227, 1999. Blood pressure was identified as astrong predictor in this study, but the predictive capacity wasincreased by the inclusion of women with chronic hypertension, a knownrisk factor for PE.

All the low risk women who volunteered for the study during the courseof the clinical trial formed the control group; this had the advantagethat the samples from all three groups were similarly treated and storedfor an identical period, but led to a significant difference inethnicity between the pre-eclamptic and low risk groups. We are notaware of any evidence in the literature to suggest any ethnic variationin the markers of oxidative stress, placental or endothelial functionstudied, although most studies do not consider ethnicity. There was alsono evidence from the statistical analysis performed in this study tosuggest that ethnicity contributed to any of the differences observed.

In conclusion, the data reports gestational trends in a wide range ofmarkers associated with PE. Our investigation has shown early andselective changes in markers of oxidative stress, lipids and some makersof placental dysfunction suggesting that these may play a role in theaetiology of the disease. Since abnormal profiles were evident severalweeks before the clinical onset of PE, we were able to identifycombinations of markers that have the potential to identify women whowill later develop PE.

All documents cited herein are incorporated by reference.

TABLE 7 Baseline characteristics in low and high- risk women accordingto clinical outcomes. Low risk High risk High risk AGA SGA PE N 27  17   21   Median Age (years) 31.9 30.8 29.9 (IQR) (30.6-34.1)(23.8-33.4) (27.5-34.9) Smokers 0  3 (17%) 1 (5%) Median body mass 23.022.9 27.0 index (kg/m²) (IQR) (21.9-24.9) (21.5-25.8) (23.5-32.5) Parity≧1 6 (22%) 6 (33%) 15 (71%) 24 week Doppler waveform analysis MedianResistance Index 0.47 0.63 0.72 (IQR) (0.44-0.55) (0.61-0.69)(0.62-0.79) Unilateral notch 0  5 (28%)  3 (14%) Bilateral notch 0  13(72%)  18 (86%) Low risk women with appropriate for gestational agedeliveries (AGA), high risk women who delivered SGA (small forgestational age) infants and high risk women who developed pre-eclampsia(PE).

TABLE 8 Perinatal characteristics in low and high- risk women accordingto clinical outcomes. Low risk High risk High risk AGA SGA PE N 27 17 21 Median systolic blood 121  125  150 pressure; maximum prior to(120-130) (120-133) (150-184) delivery (mmHg) (IQR) Median diastolicblood 80 77 106 pressure; maximum prior to (70-82) (70-86) (100-118)delivery (mmHg) (IQR) Median maximum urine  0  0 855 protein excretion(mg/24 hr) (580-3010) (IQR) Median gestational age at   40.3   39.7  37.1 delivery (weeks) (IQR) (39.1-41.2) (38.3-40.6) (34.4-38.6) Medianbirthweight (grams) 3480  2700  2500  (IQR) (3340-3770) (2353-3015)(2070-2940) Median birthweight (centile) 57  5  8 (IQR) (30-82) (1-7) (2-24) Small for gestational age  0 17 (100%) 11 (52%) infants Low riskwomen with appropriate for gestational age deliveries AGA), high riskwomen who delivered SGA (small for gestational age) infants and highrisk women who developed pre-eclampsia (PE),.

TABLE 9a Prediction of PE using biochemical indices in maternal blood at20 and 24 week's gestation. ROC areas are given (with 95% confidenceintervals). Comparison is made with low risk women with normal outcome(LR) and high risk women who delivered small for gestational age infants(SGA). If confidence intervals do not include 0.5 the difference issignificant. Biochemical 20 weeks' gestation 24 week's gestation indexPE vs. LR PE vs SGA PE vs. LR PE vs SGA 8-epi-PGF_(2α) 0.62 0.53 0.550.37 (0.44, 0.81) (0.29, 0.76) (0.35, 0.75) (0.13, 0.61) HDL-cholesterol0.73 0.75 0.61 0.64 (0.57, 0.89) (0.57, 0.93) (0.41, 0.82) (0.40, 0.87)Uric acid 0.57 0.68 0.67 0.70 (0.38, 0.76) (0.48, 0.87) (0.50, 0.85)(0.52, 0.89) PAI-1/PAI-2 ratio 0.70 0.57 0.76 0.62 (0.52, 0.87) (0.36,0.78) (0.59, 0.92) (0.42, 0.83) Leptin 0.71 0.82 0.77 0.88 (0.55, 0.88)(0.67, 0.97) (0.62, 0.92) (0.76, 1.00) Placenta Growth 0.72 0.60 0.850.73 Factor (0.54, 0.91) (0.39, 0.80) (0.71, 0.99) (0.54, 0.92)

Table 9b shows comparison when risk of PE is assessed using combinationsof biochemical indices.

Combination 20 weeks' gestation 24 weeks' gestation of indices PE vs. LRPE vs SGA PE vs LR PE vs SGA Log_(e)P1GF-3.0 0.81 0.61 0.95 0.76{PAI-1/PAI-2 (0.65, 0.97) (0.39, 0.83) (0.87, 1.00) (0.57, 0.96) ratio}PAI-2 * P1GF 0.80 0.76 0.89 0.83 (0.63, 0.97) (0.58, 0.94) (0.78, 1.00)(0.68, 0.99) Leptin/P1GF 0.80 0.76 0.89 0.83 (0.63, 0.97) (0.58, 0.94)(0.78, 1.00) (0.68, 0.99)

TABLE 10 Sensitivity and specificity (95% Confidence Intervals) for twothreshold values calculated from three identified formulae for theprediction of PE. Threshold Formula values Sensitivity Specificityloge[PlGF] − 3.0{PAI-1/ <4.5 53% 100% PAI-2 ratio} (27%, 79%) (79%,100%) <5 80%  88% (52%, 96%) (62%, 98%)  PAI-2 * PlGF <35*10³ 67% 100%(38%, 88%) (79%, 100%) <50*10³ 80%  94% (52%, 96%) (70%, 100%)leptin/PlGF ratio >0.1 67% 100% (38%, 88%) (80%, 100%) >0.05 80%  88%(52%, 96%) (64%, 99%) 

1. A kit for specific prediction of pre-eclampsia (PE) by determininglevels of two or more markers in a maternal sample, wherein the kitcomprises immunoassay reagents capable of measuring the levels of themarkers, wherein the markers are selected from the group consisting of:placenta growth factor (PlGF), plasminogen activator inhibitor-1(PAI-1), plasminogen activator inhibitor-2 (PAI-2), leptin, the ratio ofleptin/PlGF concentrations, and the ratio of PAI-1/PAI-2 concentrations.2. The kit of claim 1 wherein one of the two markers is placenta growthfactor (PlGF).
 3. The kit of claim 2 wherein one of the two markers isplasminogen activator inhibitor-2 (PAI-2).
 4. The kit of claim 2 whereinone of the two markers is the ratio of leptin/PlGF concentrations. 5.The kit of claim 2 wherein one of the two markers is the ratio ofPAI-1/PAI-2 concentrations.
 6. The kit of claim 2 wherein one of the twomarkers is leptin.
 7. The kit of claim 6 wherein a ratio of leptin/PlGFconcentration is determined.
 8. The kit of claim 1 wherein theimmunoassay reagents are selected from the group consisting of: enzymelinked immunoassay reagents, RIA reagents, and reagents for Westernblotting.
 9. The kit of claim 1 wherein the immunoassay reagents areenzyme linked immunoassay reagents.
 10. The kit of claim 1 wherein theimmunoassay reagents are RIA reagents.
 11. The kit of claim 1 whereinthe immunoassay reagents are reagents for Western blotting.
 12. The kitof claim 2 wherein the immunoassay reagents are selected from the groupconsisting of: enzyme linked immunoassay reagents, RIA reagents, andreagents for Western blotting.
 13. The kit of claim 3 wherein theimmunoassay reagents are selected from the group consisting of: enzymelinked immunoassay reagents, RIA reagents, and reagents for Westernblotting.
 14. The kit of claim 4 wherein the immunoassay reagents areselected from the group consisting of: enzyme linked immunoassayreagents, RIA reagents, and reagents for Western blotting.
 15. The kitof claim 5 wherein the immunoassay reagents are selected from the groupconsisting of: enzyme linked immunoassay reagents, RIA reagents, andreagents for Western blotting.
 16. The kit of claim 6 wherein theimmunoassay reagents are selected from the group consisting of: enzymelinked immunoassay reagents, RIA reagents, and reagents for Westernblotting.
 17. The kit of claim 7 wherein the immunoassay reagents areselected from the group consisting of: enzyme linked immunoassayreagents, RIA reagents, and reagents for Western blotting.